Student Theses and Dissertations
Date of Award
2024
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
RU Laboratory
Chen Laboratory
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed in the apical membrane of epithelial tissues. Alterations in CFTR that disrupt activity cause cystic fibrosis, a fatal disease which is characterized by systemic salt and fluid dysregulation. By contrast,hyperactivation of CFTR is central to pathogenesis in secretory diarrhea and autosomal dominant polycystic kidney disease.Electrophysiological properties of CFTR have been analyzed for decades. The structure of CFTR,determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Opening of CFTR’s pore is regulated by phosphorylation- and ATP-dependent dimerization of its two nucleotide-binding domains (NBDs), but how the dimerization process is coupled to pore opening remains contested.Whereas the binding sites for therapeutic potentiators of CFTR gating are known, the mechanism by which they increase channel open probability is not understood. In this thesis, I combined ensemble functional measurements, single-molecule fluorescence resonance energy transfer imaging, electrophysiology, electron microscopy and kinetic simulations to study human CFTR gating and conduction, as well as how these processes are altered by disease mutations and pharmacological modulation. I first established a model for gating by wild-type CFTR. I found that CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization and weaken coupling of the dimerization process to opening of the pore. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while the NBDs are dimerized. A second modulator, elexacaftor, was found to act additively with GLPG1837 or ivacaftor to further increase open probability of both the wild-type CFTR and disease-causing variants. The combined effects of elexacaftor andGLPG1837 stabilize the open pore regardless of nucleotide state in the NBDs and thereby decouplepore opening from the ATP hydrolysis cycle. Despite determination of CFTR structures, the ion conduction path remains elusive. Here, a density consistent with dehydrated chloride is directly observed within the pore of CFTR. Substitution of residues proximal to the putative chloride altered the conductance and selectivity properties of the pore. The inhibitor CFTRinh-172 was found to act by direct occlusion of this selectivity filter. Finally, knowledge of CFTR structure and mechanism was applied for structure-based discovery of small-molecule modulators that allosterically potentiate or inhibit CFTR gating. These molecules represent potential leads for drug development and demonstrate the feasibility of large-scale docking for ion channel drug discovery.
Recommended Citation
Levring, Jesper Bøegh, "Single-Molecule Studies of CFTR Gating and Pharmacology" (2024). Student Theses and Dissertations. 764.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/764
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy